1. Field of the Invention
The present invention relates to a fluorescence reader which detects fluorescence from a sample present on a carrier or in a solution.
2. Description of the Related Art
This type of reader includes a reader for measuring fluorescence of a so-called DNA chip or DNA micro array in which a complementary sequence (hereinafter referred to as a probe) is constituted in a solid phase on a carrier of glass, silicon, or plastic especially in a part of a peculiar sequence of a nucleic acid which is a detection target. In this reader, each probe is labeled with the fluorescence to measure the fluorescence. However, it is known that the quantity of fluorescence to be detected here is very small as compared with that of fluorescence for another cell or tissue which is a detection object.
Therefore, the quantity of fluorescence needs to be detected with high sensitivity. For this, the principle of a fluorescence reader is roughly divided into:                (1) a confocal/photomultiplier tube/scanning system; and        (2) a cooling CCD system. These have the following characteristics.        
(1) This system of a fluorescence reader carries out scanning by a confocal laser system, and is used mainly in an application of detecting a coupling reaction of the nucleic acid on the glass carrier. In the confocal system, noise by a disturbance light is removed, and high measurement performance is realized, but the focal depth is very shallow. The focal depth of a general confocal laser system depends on the NA of an objective lens, and is obtained by the following equation.Focal depth=(0.6×wavelength)/(NA)2
For example, when a laser having a wavelength of 632 nm is used to use a lens having an NA of 0.3, the focal depth is about 4 μm from the above equation. In an optical system having such a narrow depth, when the fluorescence is focused, clear fluorescent photometry is obtained without any noise. However, when a focal point deviates, the correct quantity of fluorescence cannot be measured. When the fluorescence is focused inside glass, self fluorescence of glass is picked up, and the background brightens. Therefore, the fluorescence is easily influenced by distortion, deflection, and the like on the side of a chip or array, and there is a fear that a uniform fluorescent screen image is not easily obtained with respect to a chip or array front plane.
Moreover, in this type of latest reader, an auto-focusing function which is tuned in the scanning is taken in, and an improvement is made so as to obtain the uniform fluorescent screen image, but there are disadvantages of enlargement and price rise of the reader.
(2) In this type of fluorescence reader, a halogen lamp is used in a light source, and a cooling CCD is used. The reader is used in the application of detecting the coupling reaction of the nucleic acid on the micro array of the glass carrier prepared using a micro array system. In this system, as compared with the system (1), a broad range can be measured at once, and a read time is short. When an exposure time is changed, samples intense to weak in the fluorescence can broadly be handled. Furthermore, there are characteristics that a filter for an exciting light is changed, and accordingly the exciting light is obtained in accordance with a fluorescent material for use.
However, in actual measurement, sensitivity of principle is inferior to that of the system (1), and there is a disadvantage that the system is insufficient in SNP dyping or gene expression frequency analysis measurement which requires high-sensitivity analysis.
(1). At present, for the chip or micro array for use mainly in a field of molecular biology, the materials of the carrier are various such as a glass plate, silicon plate, and porous filter formed of nylon or nitrocellulose. In any of these materials, subtle distortion, deflection, and self fluorescence exist. Therefore, when a very small amount of fluorescence is to be measured on the surfaces of these materials, in the above-described conventional art, uniform fluorescent photometry cannot be performed with respect to the whole measurement surface because of the distortion, and the like.
(2). Prior arts described above in (1), (2) have problems that (1) since the confocal laser system is used, exact measurement is possible at the time of focusing, but read sensitivity drops at the time of non-focusing, and that (2) the read sensitivity is low in the CCD system because of a sensitivity difference and photometry range difference between the photomultiplier tube and CCD, respectively. In general, an exciting light intensity needs to be raised in order to detect the fluorescence with the high sensitivity, but especially when the fluorescent material is an organic material, there is a problem that the material is remarkably deteriorated with an increase of the exciting light intensity.
(3). In order to overcome the problems (1), (2) described above in (2), in the latest technology, attempts have been made to adopt an auto focus system, or to increase the number of applied laser beams. Accordingly, control measurement is performed to make corrections. However, these measures tend to enlarge the reader and to raise prices. Moreover, the carrier has protrusions such as partition walls depending on the shape of the carrier, and the optical systems such as an auto focus cannot be added in some case.
(4). In the conventional reader, when the focal point is moved only by several micrometers, a fluorescence read amount largely changes, the amount of noises largely increases/decreases, and it is not thus easy to set measurement conditions.
An object of the present invention is to provide a fluorescence reader in which the uniform fluorescence measurement is carried out regardless of a state of the measurement object, the very small amount of fluorescence can be detected with the high sensitivity, the setting of the measurement conditions is facilitated, and miniaturization of the reader is achieved.